Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants

Significance Immunity induced by the first-generation COVID-19 vaccines may not provide effective and durable protection, either due to waning immunity or due to poor antibody cross-reactivity to new variants. Typically, T cells recognize conserved nonmutable viral epitopes and development of T cell–based vaccines might provide broad immunity to SARS-CoV-2 variants. In this study, we show that adjuvanted spike protein–based experimental vaccines elicited potent respiratory or systemic CD4 and CD8 T cell memory and protected against SARS-CoV-2, in the absence of virus-neutralizing antibodies. Thus, development of T cell–based vaccines might be key to protect against antibody-escape SARS-CoV-2 variants that can potentially overcome immunity induced by current vaccines.


Supplemental Figure 3. Functional Polarization of Effector CD8 T Cells in Vaccinated Mice.
Cohorts of C57BL/6 mice were vaccinated twice with recombinant SARS-CoV-2 spike protein formulated in ADJ+CpG or ADJ+GLA by the IN or the SQ route. At day 8 after booster vaccination, cytokine production by K b /S525-specific CD8 T cells were analyzed by intracellular cytokine staining.
Lung cells or splenocytes were stimulated with S525 peptide, as described in   Anti-CD8 Anti-CD4

Tissue processing and Flow cytometry
Spleens and lungs were processed into single cell suspensions using mechanical digestion and standard collagenase-based methods, as previously described (1). To stain for surface markers, single-cell suspensions were first stained for viability with Ghost Dye™ Red 780 (Tonbo Biosciences, stained with antibodies and tetramers diluted in Brilliant Stain Buffer (BSB, BD Biosciences) for 60 minutes at 4C, and fixed with 2% paraformaldehyde. All samples were acquired on LSRFortessa (BD Biosciences) and analyzed with FlowJo V.10 software (TreeStar, Ashland, OR).

Intracellular staining for transcription factors
To stain for intracellular factors directly ex vivo, single-cell suspensions were stained for viability as above. Next, samples were stained with antibodies and tetramers diluted in BSB (BD Biosciences), which were then fixed, permeabilized and subsequently stained for using the transcription factors staining kit (eBioscience) with the antibodies (indicated in Supplemental Table 1) diluted in eBioscience Perm Wash buffer. All samples were acquired on LSRFortessa (BD Biosciences) and analyzed with FlowJo V.10 software (TreeStar, Ashland, OR).
Samples were stained with antibodies corresponding to cytokines indicated in Supplemental Table 1 in perm wash buffer. All the staining procedures were performed on ice.

Cells and Viruses
African Green Monkey Kidney Cells (Vero), and Human Embryonic Kidney 293T (HEK293T) cells were obtained from ATCC (ATCC; Manassas, VA, USA). SARS-CoV-2 USA-WA1/2020 (WA strain) and hCoV-19/South Africa/KRISP-EC-K005321/2020 (SA strain) viruses used in these studies were obtained from BEI resources (NR-52281, NR-55282 respectively), and propagated in Vero cells. Adenovirus Serotype 5, co-expressing recombinant human ACE2 (Ad5-hAce2) was obtained from BEI resources (NR-52390) and propagated in HEK293T cells and purified as previously described (2). SARS-CoV-2 WA strain viral titrations were performed by a focus forming unit assay with some modifications (3). SARS-CoV-2 SA strain viral titrations were performed similarly, but cells were incubated with virus for 5 days, fixed, and plaque forming units were calculated from observing cytopathic effects under a light microscope. SARS-CoV-2, isolate USA-WA1/2020 (lineage A), or isolate SA/2020 (lineage B.1.351) was propagated and titrated on Vero E6 cells. Serial dilutions were made from serum (1:20-1:14,580; 3-fold) and BAL (Undiluted-1:80; 2-fold) in serum-free Opti-MEM media and incubated with 100 PFU per well of SARS-CoV-2 isolates for 60 mins at 37°C and transferred into wells pre-seeded with Vero E6 cells. Plates were incubated at 37°C for 3-4 days before scoring for the cytopathic effect. Neutralization titer was calculated as the reciprocal of the highest dilution at which virus neutralization occurred.

Viral challenge
For all challenge studies, mice were euthanized on day 5 post challenge and lungs collected for viral titers (left lobe), histology, (a section of the inferior lobe) and T-cell analysis (remainder of lung tissue). For virus titration, lungs were weighed, then homogenized in Opti-MEM media containing 3% FBS via bead beating, clarified by centrifugation and titrated as described above.
Lung sections for histology were taken from uninflated lungs and fixed in 10% neutral buffered formalin, sectioned, and stained with Hematoxylin and Eosin (H&E) by conventional methods.